Methods and apparatus for forming 2-dimensional drop arrays

ABSTRACT

Certain embodiments are directed to finite step emulsification device and/or methods that combine finite step emulsification with gradients of confinement for the formation of a  2 D monolayer array of droplets with low size dispersion.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser.16/385,029, filed Apr. 16, 2019, which claims the benefit of priority toU.S. application Ser. 62/658,172 filed Apr. 16, 2018. The contents ofeach of the referenced applications are incorporated herein by referencein their entireties.

BACKGROUND OF THE INVENTION A. Field of the Invention

Embodiments generally concern microfluidic devices. Particularembodiments are directed to microfluidic devices for production ofuniform droplet arrays.

B. Description of Related Art

A primary cost driver of a microfluidic system can be a consumablemicrofluidic circuit portion. In certain aspects each microfluidiccircuit can have many very small features that need to be identical insize within about a 1-2 micron variance. Therefore, the manufacturingprocess needs to be well controlled. Given the microfluidic circuits areconsumable, the manufacturing process for the microfluidic circuitsneeds to be operational for large scale manufacturing.

Masters for molding can be produced generally in two ways. The first isby micro-milling. Micro-milling is a reductive manufacturing processwhere a computer controlled rotating cutter removes material from asolid block of steel, brass, aluminum or other material. Practicallyspeaking micro-milling can produce features on the order of 10 μm andhold tolerances on the order of +/−1 μm. However, to achieve this rangeof tolerances you must control cutter wear, and/or vibration andtemperature of stock and cutter. These things become more of a problemas the number of features increases and the micro-milling timeincreases. For example, producing a microfluidic chip master containing32 microchannels with a tolerance of +/−1 μm using micro-milling cannotbe currently done commercially.

Another way to produce a master is to use a lithographic process to etchsilicon. The features of the etched silicon are then transferred tometal through an electroplating process. This process is capable ofholding +/−1 μm regardless of the number of features because it is notsusceptible to factors like tool wear, vibration, and thermalconditions. Standard lithographic methods are limited to features thatcan be mastered to near 90-degree walls. A ramp or angled region cannotbe produced using standard lithography. A ramp can be approximated inlithography by an infinite number of 90-degree steps, but this is costprohibitive. Numerous etching passes are needed to result in a ramp-likecascade of steps. Such a step-down process for manufacturing a ramp-likestructure would significantly impact the cost of mastering.

There remains a need for a microfluidic circuit design and method ofmanufacturing that can be used to cost effectively producetwo-dimensional (2D) droplet arrays that have a low drop sizedispersion.

SUMMARY OF THE INVENTION

Certain embodiments provide a solution to the manufacturing problemsassociated with microfluidic devices for forming substantially uniformdrop arrays. In particular, a microfluidic device described herein isdesigned so that it can be manufactured efficiently and cost effectivelyby micro-milling and lithographic production methods. By way of example,the inventors have designed a device that performs a process for forminglow size dispersion 2D droplet arrays. The device uses a microfluidiccircuit having a nozzle, a step emulsification region, ramp region, andan imaging or array region. The device and process produce a 2D dropletarray having low size dispersion for a more cost effective and robustanalysis.

Embodiments are directed to finite step emulsification combined withgradients of confinement for the formation of a 2D monolayer array ofdroplets with low size dispersion. In certain aspects unstacked 2Dmonolayer array of droplets with <3% size dispersion can be produced.Certain embodiments are directed to a microfluidic device configured toform a low size dispersion 2D monolayer array.

Certain embodiments are directed to microfluidic circuits comprising:(a) a drop forming region comprising a channel or nozzle having an inletfor receiving a sample, the channel having a constant cross-sectionalarea, and an outlet that opens into a step region, the channel or nozzlebeing 10 to 1000 μm long, and a rectangular cross-section with a heightof 1 to 20 μm and a width of 2 to 60 μm; (b) the step region havinglength extending from the nozzle of 15 to 500 μm, a top and a bottomwith a height of between 5 to 80 μm, the top or bottom beingsubstantially continuous with the top or bottom, respectively, of thechannel or nozzle and the bottom or top being offset from the bottom ortop of the channel or nozzle and substantially parallel with the bottomor floor, or top or ceiling of the channel or nozzle, forming a stepregion configured for finite step emulsification; (c) a ramp regionhaving an angle of between 10 and 80 degrees relative to the floor orceiling of the step region (the ramp can be positioned above or belowthe step region depending on design choice) and a length of 10 μm to1000 μm, the ramp region starts at the end of the step region andterminates in an imaging region (in certain aspects the ramp starts at aheight of 5 to 80 μm (determined by the step region height) andincreases to a height of 15 or 110 μm over a horizontal length that isdetermined by the angle of the ramp); and (d) the imaging region isconfigured to collect drops as a two-dimensional array and to providefor imaging and evaluation of the two-dimensional drop array. Themicrofluidic circuit can be fluidly connected to a sample reservoir viaa flow path that is configured to provide sample to be received by eachnozzle inlet. In certain aspects the flow path is a channel havingcross-section with a height of 10 to 200 μm and a width of 10 to 200 μm.The microfluidic circuit can be fluidly connected to a waste reservoirconfigured to receive sample droplets after analysis. In certain aspectsthe ratio of the nozzle height to step region height is less that 1:3,in particular 1:2.8. In other aspects the ramp angle is 15 to 45degrees, particularly 30 degrees. In certain aspects the microfluidiccircuit is made of a thermoplastic polymer. In particular aspects themicrofluidic circuit is a cyclic olefin polymer or copolymer,polycarbonate, or poly (methyl methacrylate) (PMMA).

In other embodiments the microfluidic circuit can be part of amicrofluidic analysis system that is operatively position or connectedto an image analysis device. The image analysis device can be configuredto detect and analyze fluorescence of droplets in the imaging region ofthe microfluidic circuit. The imaging device can comprise a camera and amicrocontroller.

Certain embodiments are directed to methods of finite stepemulsification comprising flowing a dispersed phase through a nozzlehaving a constant cross-sectional area into a confined step regioncontaining a continuous phase and forming constrained droplets,providing a ramp region having an increasing cross-section area throughwhich the constrained droplets flow forming an unconstrained droplet,where the unconstrained droplets are deposited in an array region andform a two dimensional droplet array having a low size dispersion. Incertain aspects the dispersed phase is an aqueous sample. The aqueoussample can be a biological or environmental sample. In other aspects thecontinuous is immiscible with the disperse phase. The continuous phasecan be a fluorocarbon.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be embodimentsof the invention that are applicable to all aspects of the invention. Itis contemplated that any embodiment discussed herein can be implementedwith respect to any method or composition of the invention, and viceversa.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

The term “about” or “approximately” are defined as being close to asunderstood by one of ordinary skill in the art. In one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The term “substantially” and its variations are defined to includeranges within 10%, within 5%, within 1%, or within 0.5%.

The terms “inhibiting” or “reducing” or “preventing” or any variation ofthese terms includes any measurable decrease or complete inhibition toachieve a desired result.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The terms “wt. %,” “vol. %,” or “mol. %” refers to a weight, volume, ormolar percentage of a component, respectively, based on the totalweight, the total volume, or the total moles of material that includesthe component. In a non-limiting example, 10 moles of component in 100moles of the material is 10 mol. % of component.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The compositions and methods of making and using the same of the presentinvention can “comprise,” “consist essentially of,” or “consist of”particular ingredients, components, blends, method steps, etc.,disclosed throughout the specification.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification embodiments presented herein.

FIG. 1 is an overhead illustration of one example of a microfluidicdevice designed to produce a 2D monolayer array having a low sizedispersion or uniform drop array.

FIG. 2 is a cross section view of one example of a microfluidic devicethat is produced as a first micro-mill component and a secondlithographic component that can be aligned to form a fully integratedmicrofluidic device.

FIG. 3 illustrates four general regions of the microfluidic design:region 1=nozzle, region 2=finite step emulsification region, region3=ramp region, region 4=imaging or array region.

FIG. 4 illustrates one example of drop formation as a sample movesthrough a microfluidic device.

FIG. 5 illustrates some of the issues that can be encountered usingcurrent designs to produce small droplet arrays.

FIG. 6 is a photograph of an array produced using methods describedherein. The image has a droplet size dispersion of 2.3% (1456 dropletswith a coefficient of variation equal to 2.32%).

DETAILED DESCRIPTION OF THE INVENTION

In conducting 2-dimensional (2D) array analysis it is beneficial tomaximize the number of tests that can be run on a given patient sampleand minimize the cost of consumables used for performing the analysis.In certain aspects, a patient sample is ideally assessed for the largestnumber of relevant antibiotics, or other compounds/characteristics, todetermine the susceptibility of, for example, one or more microbes inthe patient sample. To maximize the number of relevant queries, apatient sample can be split into a plurality of equal volumes. Eachvolume is then mixed individually with a test substance, e.g.,antibiotic(s), and then introduce into a microfluidic circuit wheredroplets of that individual volume are then created and analyzed. Anindividual volume of patient sample can be process on a microfluidiccircuit separate from other volumes of the patient sample. In certainaspects 1, 2, 4, 8, 16, 24, 32, 40, 48 or more (including all values andranges therein) microfluidic circuits can be employed.

FIG. 1 provides an overhead view of an example of one embodiment of amicrofluidic circuit. The circuit comprising the drop forming regionincluding the channels or nozzles 102 fluidly connected to step region104, that is fluidly connected to ramp region 106 which is in turnconnected to imaging area or region 108. Channels 102 are fluidlyconnected to sample source or reservoir 112 via flow path 101. Incertain aspects gutters 110 can be included in the design as well aswaste reservoir 114.

FIG. 2 depicts cross section AA. Flow path 201 is fluidly connected tochannel or nozzle 202. Channel or nozzle 202 flows into step region 204.Step region 204 flows into ramp region 206. Ramp region 206 then flowsinto imaging region 208. The device can be manufactured in two pieces(i) micro-milled portion and (ii) a lithographically produced portion,which can be fixed together using methods well known in the art to forma microfluidic circuit.

A. Microfluidic Circuits

In certain embodiments a primary cost driver of a microfluidic systemcan be a consumable microfluidic circuit portion or microfluidicdevice/chip/disposable containing a microfluidic circuit. In certainaspects each microfluidic circuit can have many very small features thatneed to be identical in size within about a 1-2 micron variance.Therefore, the manufacturing process needs to be well controlled. Giventhe microfluidic circuits are part of a consumable, the manufacturingprocess for the microfluidic circuits needs to be operational for costeffective, large scale manufacturing. In certain aspects themanufacturing process for the consumable having a microfluidic circuitcan be compression injection molding. Like injection molding,compression injection molding replicates the features of a master into aplastic part. The difference is that the compression injection moldingallows for much higher precision and smaller features. The part qualityproduced from compression injection molding is only as good as themaster used to form the features. Therefore, a high-quality master isdesirable.

Masters can be produced generally in two ways micro-milling andlithography. Micro-milling is a reductive manufacturing process where acomputer controlled rotating cutter removes material from a solid blockof steel, brass, aluminum or other material. Practically speakingmicro-milling can produce features on the order of 10 μm and holdtolerances on the order of +/−1 μm. However, to achieve this range oftolerances you must control cutter wear, and/or vibration andtemperature of stock and cutter. These things become more of a problemas the number of features increases and the micro-milling timeincreases. For example, producing a 32-circuit microfluidic chip masterwith a tolerance of +/−1 μm using micro-milling cannot be currently donecommercially. The fewer the microfluidic circuits per chip the greaterthe number of chips needed resulting in increased cost.

Lithography is a process for etching silicon. The features of the etchedsilicon are then transferred to metal through an electroplating process.This process is capable of holding +/−1 μm regardless of the number offeatures because it is not susceptible to factors like tool wear,vibration, and thermal conditions. Lithographic methods are capable ofproducing chips having numerous circuits in a chip, limiting the numberof consumables to be used. However, standard lithographic methods arelimited to features that can be mastered to with near 90-degree walls.In certain embodiments a microfluidic circuit described herein uses aramp that can be, but is not limited to, between 10 and 50 degrees or soin the microfluidic circuit design. A ramp can be approximated inlithography by an infinite number of 90-degree steps, but this would becost prohibitive. A step-down process to manufacture a ramp structurewould significantly impact the cost of mastering.

In certain embodiments a microfluidic circuit can be manufactured usinga two-piece manufacturing process with a first component being generatedusing compression injection molding or an analogous process where alithographically generated master is used to form small channels and asingle step region, which are structures that are easily and costeffectively produced using lithography. A second component can begenerated using compression injection molding or an analogous processusing a micro-milled master for those structures that are structuresthat are cost effectively produced using micro-milling, which includesstructures such as but not limited to the ramp region and imagingregion. These two components can be aligned and bonded to form amicrofluidic circuit as described herein.

The alignment precision between a drop formation component (e.g., ii ofFIG. 2 , region 1 and region 2 of FIG. 3 ) and an array formationcomponent (e.g., i of FIG. 2 , region 3 and region 4 of FIG. 3 ) can beon the order of +/−50 μm. The alignment need not be tightly controlledbecause the position of the ramp with respect to the droplet formationfeatures does require 1 to 2 μm precision in order to maintainconsistent droplet size. Lithography can be used to form the features ofthe design that are critical to droplet formation and micro-milling canbe used to form the non-critical features relative to drop formation,which are then bonded together to make a microfluidic device having anarray of microfluidic nozzles and related circuit that are allessentially identical from a droplet formation perspective. The dropletsare formed in a portion of the device produced with a high degree ofprecision (+/−1 to 2 μm) and the drops finished and processed in aportion of the device that can be produced with a lesser degree ofprecision (+/−5 to 30 μm).

A device that requires a ramped portion to form the drops (nozzle/rampconfiguration) would suffer from issues of alignment because the ramp isintegral in forming the drops and must be positioned with much greaterprecision to form the droplets with a low dispersion. If the ramp wasnot nearly perfectly aligned with the outlet of the small channels inthe nozzle/ramp configuration the droplet size and consistency would beaffected. Therefore, a nozzle outlet directly feeding a ramp regionwould likely see significant variation from part to part if they triedto have more than 1, let alone up to 32, microfluidic circuits in onepart. A nozzle/ramp (as contrasted with a nozzle/step/rampconfiguration) would be difficult to impossible to produce costeffectively at the tolerances needed.

B. Low Size Dispersion Droplet Emulsions

There are multiple ways to form low size dispersion droplet emulsions.The most common method is to use a two-phase flow system. This methodtypically uses the shear stress imparted by a flowing continuous phaseto form droplets from a flowing dispersed phase. As the name implies,this method requires both phases to be flowing. Additionally, tomaintain low droplet size dispersion one of the flow rates (eithercontinuous or dispersed) requires precision control. Due to the need fortwo different flow rates, this method requires a more complex system toemploy.

Another way to form low size dispersion droplet emulsions is to usesingle phase flow techniques. In single phase flow systems, thedispersed phase flows while the continuous phase remains static. Thesesystems may use changes in Laplace pressure or buoyancy to inducedroplet formation. Although the system complexity is lower with singlephase flow compared to two-phase flow, there are other challenges withthe single flow method that need to be addressed if the goal is to forma 2-D monolayer array of low size dispersion droplets (see FIG. 5 for afew examples).

One of the first methods to use the single-phase flow technique was stepemulsification. Step emulsification can be embodied in two general ways.The first embodiment can be considered as a channel carrying a dispersedphase that is introduced into a semi-infinite step containing a staticcontinuous phase. A semi-infinite step is defined as a step that issignificantly greater than 1× the unconfined formed droplet diameter. Inthis scenario, a droplet will be created by gradients in Laplacepressure. After formation, the droplet may rise, fall, or remain at thenozzle exit depending on the relative buoyancy of the dispersed phasecompared to the continuous phase. If the buoyancy is mismatched, theformed droplets will float or sink away from the nozzle until they reachthe semi-infinite wall. Droplets formed in semi-infinite step systemshave excellent size dispersion due to the consistent environment at theoutlet of the nozzle. However, if the droplet production rate is highenough, the droplets will begin to stack at the semi-infinite wall and a2D monolayer will not be achieved until buoyancy forces the droplets tounstack. Buoyancy is a relatively weak force on picoliter droplets, sounstacking takes a very long time. Additionally, if the stacking issevere, the stacked droplets will eventually crowd at the outlet of thenozzle and obstruct the formation of new droplets. This crowding createsan inconsistent environment at the outlet of the nozzle, and thereforeincreases the size dispersion. If the buoyancy between dispersed andcontinuous phases is matched, the droplets will crowd at the outlet ofthe nozzle and obstruct the formation of new droplets. This crowdingincreases the droplet size dispersion for reasons mentioned above. Thecrowded droplets are also free to stack due to the lack of confinementand a 2D monolayer will not be achieved.

The second embodiment of step emulsification can be considered as achannel carrying a dispersed phase (e.g., an aqueous phase) that isintroduced into a finite step containing a static continuous phase(e.g., an oil phase). A finite step is defined as a step that issignificantly less than 1× the unconfined droplet diameter. In thisembodiment the droplet remains compressed after formation, so buoyancydoes not play a roll. This embodiment is favorable to the first becauseit ensures the formed droplets create a 2D monolayer. But because thereis no mechanism to clear the formed droplets from the nozzle, thedroplets crowd at the outlet of the nozzle and obstruct the formation ofnew droplets, increasing the size dispersion of the 2D monolayer arrayformed by a finite step emulsion.

The methods described herein combine finite step emulsification with afeature to maintain a consistent environment at the outlet of thenozzle. The result is a 2D monolayer array of droplets with low sizedispersion (see FIG. 6 for an example).

A research group headed by Charles Baroud from the Ecole Polytechniquehas shown a method for creating relatively large low size dispersion 2Dmonolayer droplet arrays using gradients of confinement. Using theirmethod, they have been able to achieve size dispersions as low as 3% inthese relatively large droplets. The method propose here differs fromBaroud in that it designed to form much smaller small droplets with asize dispersion significantly better than many of other single-phaseflow systems forming a 2D monolayer array of droplets. The currentdevice and methods produce a much smaller droplet than the Baroud methodusing a microfluidic circuit that is better designed for high volumearrays. The microfluidic circuit design combines four regions (see FIG.3 and FIG. 4 ) to create a consistent environment at the outlet of thenozzle during droplet production that results in a 2D monolayer array ofdroplets with low size dispersion.

C. Microfluidic Device

In certain embodiments a microfluidic device is designed to produce a 2Ddroplet array with low size dispersion. In statistics, dispersion orvariability is the extent to which a distribution is stretched orsqueezed. Common examples of measures of statistical dispersion are thevariance, standard deviation, and interquartile range. As used hereinlow size dispersion refers to dispersion of less than or about 6, 5, 4,3, 2, or 1%, including values and ranges there between. In certainaspects low size dispersion refers to a dispersion of less than 3%. Inparticular aspects the droplet size dispersion is about 2.1 to 2.5%, or2.3%.

The methods and devices described herein create a 2D monolayer array ofdroplets with low size dispersion by using four processing regions. Incertain aspects a device or portion of a device that comprises all fourregions operatively coupled as described below is referred to as amicrofluidic circuit. A microfluidic device can include 1, 2, 4, 8, 16,32, 64, 128 or more (including all values and ranges there between)channel regions or nozzles coupled to a second, third and fourth region.In certain aspects all or some of the nozzles will be in fluid contactwith a common step, ramp, and array region.

The first processing region is the channel region or nozzle thatintroduces the dispersed phase. This region has constant cross-sectionalarea and has the highest surface energy. In certain aspects the crosssectional area can be 1, 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950 to 1000 μm², includingall values and ranges there between.

The second processing region is the finite emulsion step region. Thisregion has constant cross-sectional area and the second highest surfaceenergy. These two regions (channel/nozzle and finite step) are wheredroplet formation occurs. The finite step region has a second functionwhich is to hold the formed compressed droplet in place until thechannel introduces more of the dispersed phase into the finite stepregion. This additional dispersed phase nudges the compressed formeddroplet that is held in the finite region into the third region. (seefor example FIG. 4 )

The third processing region is the ramp region. This region hasincreasing cross sectional area and its surface energy ranges fromsecond highest at the upstream extreme to third highest in thedownstream extreme. When the compressed formed droplet enters thisregion, the droplet will progressively lower its surface energyproportionally by decompressing in the direction of increasing crosssectional area. This change in surface energy acts to propel the dropletthrough the ramp region downstream into the fourth processing region.This ramp region prevents droplets from the fourth region fromback-stacking into the second step region so that the environment in thesecond step region is consistent and controlled for consistent dropletformation.

The fourth processing region is the collection region where the 2Dmonolayer is formed. Here the fully formed droplets are at the lowestsurface energy. The surface energy in this region is also constantbecause the cross-sectional area is constant. The monolayer array ofdroplets grows over time as droplets are added to the region. These fourregions combine to create a consistent environment during dropletproduction that results in a 2D monolayer array of droplets with lowsize dispersion.

The microfluidic circuit comprising these four regions can be coupled toa sample source through a common flow pathway. The common flow pathwaywill have an inlet at the proximal end of the path and an outlet orplurality of outlets along the path connected to channelregions/nozzles.

D. Methods of Array Formation

In certain aspects the droplets are in an immiscible fluid. Upon mixingof a target-containing solution and the immiscible fluid, they formphases—an aqueous drop or partition, which holds the target material insolution, and a non-aqueous or continuous phase made up of theimmiscible fluid. The immiscible fluid can be a fluorocarbon comprisinga fluorosurfactant or hydrocarbon oils such as mineral oil, or siliconeoils. In particular aspects the droplets can be between 0.1 pL and 10nL. In a further aspect the droplets are at least, at most, or about0.1. 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pL to 200,300, 400, 500, 600, 700, 800, 900, or 1000 pL, including all values andranges there between. In certain aspects the droplets are about 40 to300 pL, more particularly are or about 143 pL. In certain aspects thedroplets have a diameter of 40, 45, 50, 55, 60, 65, 70, 75, 80, to 85μm. In particular aspects the droplets are or are about 65 μm. Themethods can further comprise arranging the droplets in a 2D array. Incertain aspects the two-dimensional array is a static two-dimensionalarray.

Monitoring of the optical characteristics of the droplets can beperformed using a detector, such as a camera or the like. In certainaspects the optical characteristics include fluorescence of thedroplets. In certain aspects the monitoring of the opticalcharacteristics of the droplets further comprises illuminating with orexposing the droplets to electromagnetic radiation, such as light. Incertain aspects the electromagnetic radiation comprises an excitationwavelength that is compatible with the reporter, i.e., illuminating orirradiating a droplet with an appropriate source. In certain aspects thesource provides light including an excitation wavelength of 500, 525,550, 575, 600, 625, 650, 675, to 700 nm, including all values and rangesthere between. The source will be selected so that the electromagneticradiation excites one or more reporter, e.g., dyes or other compounds.In particular aspect the light source can be a light emitting diode(LED).

Reporters can include a “viability dye” or “reporter dye”, the viabilityor reporter dye is a moiety that detects changes in the environmentsurrounding an isolated cell due to a cell's viability, respiration, ormetabolic activity; or is a detectable protein that is expressed underspecific conditions (e.g., green fluorescent protein or luciferase). Incertain aspects a cell can be transfected or engineered to express areporter protein. The reporter can be detected using any method known inthe art appropriate to the reporter employed, for example light emissionor absorbance of a fluorophore or a colorimetric dye. In certaininstances, the signal from the reporter is detected by opticalmicroscopy, camera, or other detector/sensor as appropriate. In certainaspects the reporter is a fluorescent dye. In certain aspects thereporter is resazurin, a resazurin-based dye, or a dye that is aderivative of or structurally related to resazurin(7-Hydroxy-3H-phenoxazin-3-one 10-oxide). Resazurin is a non-toxic, cellpermeable compound that, in its oxidized state, is blue in color andvirtually non-fluorescent. When in contact with living cells, resazurinis reduced to resorufin, a compound that is red in color and highlyfluorescent and can be detected fluorimetrically or colorimetrically.Metabolic activity of viable cells continuously converts resazurin toresorufin, increasing the overall fluorescence and color of the mediasurrounding cells. A resazurin-based dye is a dye that contains aresazurin structure in addition to other modifying groups. In otheraspects a viability dye is tetrazolium, a tetrazolium-based dye, or adye that is a derivative of or structurally related to tetrazolium. Atetrazolium-based dye is a dye that contains a tetrazolium moiety andmay contain other modifying groups that do not disrupt the five memberedtetrazolium ring.

In certain aspects the incubating of the droplets is at a constanttemperature (isothermal). In other aspects the temperature can becontrolled and can be stepped or ramped up using a particular intervalor rate, such as stepping up from 25 to 37° C. or increasing at a rateof 2 to 10° C. per minute. In still other aspects temperature can bedecreased at a particular interval or rate, such as decreasing at aninterval of 5 to 10° C. or a rate of 2 to 10° C. per minute. In variousaspects the temperature(s) are in the range of 20 to 45° C., 30 to 40°C., or 35 to 38° C., including all values and ranges there between. Incertain aspects partitions are incubated at 37° C.

In particular aspects a droplet can comprise a single cell, microbe, orcellular or microbial aggregation. In a further aspect the droplet maycontain 2, 3, 4, or more cell or microbe types. The methods can furthercomprise classifying a microbe by species, genus, family, order, class,phylum, kingdom, or a combination thereof. The classification can bebased on the characteristics of one or more waveforms under one or moreconditions. In certain aspects the microbe is bacteria. Certain aspectsof the invention can include classifying the bacteria by gram-staingroup or other classification criteria recognized for microbes,including bacteria. In certain instances, a droplet may contain morethan one target type (species, genus, etc.) but that an environmentalstressor or condition may kill all but one target type, which can beidentified using its signature or waveform.

The methods can further comprise dividing the sample into at least acontrol sample and at least one test sample prior to forming a 2D array.Each test sample can be treated or processed in a manner that differsfrom the control. In certain aspects at least one test sample iscontacted with a stressor, cytotoxic, anticancer, antimicrobial compoundor condition. In certain aspects individual test samples can be exposedto different concentrations compounds or variations in conditions. Inother aspects, a test sample can be exposed to a variety oftemperatures, environments, or chemicals that may or may not alter thephenotype of the cells contained in the test sample. In certain aspectsa DCC is a pathologic or pathogenic cell, such as a cancer cell.

The methods include evaluating the sample (control and test samples)using a partition waveform (i.e., signal detected over time). In certainaspects evaluating includes comparing the partition waveform to alibrary of stored or predetermined waveforms (e.g., waveform reference).

Certain embodiments are directed to methods for detecting andcharacterizing DCCs, such as microbes or cancer cells, in a samplecomprising (a) contacting a sample comprising microbes with a reporter,e.g., a viability dye, forming a sample mixture; (b) dividing the samplemixture into at least two portions or samples that include a controlsample and at least one test sample; (d) introducing a testcompound/substance or an antimicrobial drug to the at least one testsample; (e) partitioning each of the control sample and at least onetest sample into droplets forming control sample droplets and testsample droplets, where the droplets comprise on average at most onetarget microbe or a natural aggregation of microbes; (f) incubating thedroplets over time at a specific temperature or temperatures; (g)monitoring optical characteristics of the droplets during the incubationtime, wherein the optical characteristics include the amount of opticalsignal produced by interaction of the reporter with the microbe/cell inthe droplet; (h) constructing an optical signal waveform for droplets,e.g., a partition or droplet waveform; (i) classifying the microbe/cellwithin each droplet using the partition waveform shape; and (j)comparing partition waveforms between the control sample partitionwaveforms and the test sample partition waveforms and assessing testcompound/substance or antimicrobial drug susceptibility based partitionwaveform comparison. In certain aspects a test compound can include asmall molecule, peptide, a nanoparticle, or a protein. In still otherinstances a test substance can include bacteriophage and otherengineered therapeutics. In other aspects the test compound/substancecan be a therapeutic identified as a therapy or engineered as a therapyfor other disease conditions, such as cancer (e.g., chemotherapeutic oranti-cancer compound or substance).

As used herein, the term “droplet” refers to a volume of fluid (e.g.,liquid or gas) that is a separated portion of a bulk volume. A bulkvolume may be partitioned into any suitable number (e.g., 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, etc.) of smaller volumes or droplets. Droplets may beseparated by a physical barrier or by physical forces (e.g., surfacetension, hydrophobic repulsion, etc.). A droplet may (i) reside upon asurface or (ii) be encapsulated by a fluid with which it is immiscible,such as the continuous phase of an emulsion, a gas, or a combinationthereof. A droplet is typically spherical or substantially spherical inshape but may be non-spherical. The shape of an otherwise spherical orsubstantially spherical droplet may be altered by deposition onto asurface. A droplet may be a “simple droplet” or a “compound droplet,”wherein one droplet encapsulates one or more additional smallerdroplets. The volume of a droplet and/or the average volume of a set ofdroplets provided herein is typically less than about one microliter,for example droplet volume can be about 1 μL, 0.1 μL, 10 pL, 1 pL, 100nL, 10 nL, 1 nL, 100 fL, 10 fL, 1 fL, including all values and rangesthere between. The diameter of a droplet and/or the average diameter ofa set of droplets provided herein is typically less than about onemillimeter, for example 1 mm, 100 μm, 10 μm, to 1 μm, including allvalues and ranges there between. Droplets may be formed by the devicesand methods described herein and are typically monodisperse orsubstantially monodisperse (differing by less than 6, 5, 4, 3, 2, or1%).

A test sample comprising at least one target cell can be combined with aviability or reporter dye and partitioned into droplets such that astatistically significant number of droplets contain no more than onetarget cell or aggregation of cells (some microbial species tend toaggregate into cell clusters or chains). In a preferred embodiment, aviability or reporter dye will be reduced from a non-fluorescentmolecule to a fluorescent molecule in the presence of a viable cell andthen further reduced to a non-fluorescent molecule if the redoxpotential in the droplet drops below a certain amount, typically −100mV. The fluorescent signature generated in each droplet is monitoredover time and used to identify and characterize the cell containedwithin. Further details on the processes of the invention are providedbelow.

Test Sample. Target cells in the test sample include bacteria, fungi,plant cells, animal cells, or cells from any other cellular organism.The cells may be cultured cells or cells obtained directly fromnaturally occurring sources. The cells may be obtained directly from anorganism or from a biological sample obtained from an organism, e.g.,from sputum, saliva, urine, blood, cerebrospinal fluid, seminal fluid,stool, and tissue. Any tissue or body fluid specimen. In one embodimentthe test sample includes cells that are isolated from a biologicalsample comprising a variety of other components, such as non-targetcells (background cells), viruses, proteins, and cell-free nucleicacids. The cells may be infected with a virus or another intracellularpathogen. The isolated cells may then be re-suspended in different mediathan those from which they were obtained. In one embodiment the testsample comprises cells suspended in a nutrient medium that enables themto replicate and/or remain viable. The nutrient media may be definedmedia with known quantities or all ingredients or an undefined mediawhere the nutrients are complex ingredients such yeast extract or caseinhydrolysate, which contain a mixture of many chemical species of unknownproportions, including a carbon source such as glucose, water, varioussalts, amino acids and nitrogen. In one embodiment, the target cells inthe test sample comprise pathogens and the nutrient media comprises acommonly used nutrient broth (liquid media) for culturing pathogens suchas lysogeny broth, nutrient broth or tryptic soy broth. In anyembodiment the media may be supplemented with a blood serum or syntheticserum to facilitate the growth of fastidious organisms.

Compartmentalization/Partitioning. The methods of the invention involvecombining a test sample comprising at least one target cell with aviability or reporter dye and then partitioning the test sample intodroplets such that no droplet contains more than one target cell or cellaggregates. The number of droplets can vary from hundreds to millionsdepending on the application and droplet volumes can also vary between 1pL to 100 nL depending on the application, but preferably between 25-500pL with a low size dispersion. Droplet formation occurs when an aqueousor disperse phase, the test sample in this case, is introduced into animmiscible phase, also referred to as the continuous phase, so that eachdroplet is surrounded by an immiscible carrier fluid. In one embodimentthe immiscible phase is an oil wherein the oil comprises a surfactant.In a related embodiment, the immiscible phase is a fluorocarbon oilcomprising a fluoro-surfactant. An important advantage to using afluorocarbon oil is that it is able to dissolve gases relatively welland it is biologically inert. Thus, the fluorocarbon oil used in themethods described herein comprises solubilized gases necessary for cellviability.

Reporters. A variety of reporters may be used with the systems andmethods disclosed herein. For example, the at least one small moleculemetabolic reporter can be a fluorophore, a protein labeled fluorophore,a protein comprising a photooxidizable cofactor, a protein comprisinganother intercalated fluorophore, a mitochondrial vital stain or dye, adye exhibiting at least one of a redox potential, a membrane localizingdye, a dye with energy transfer properties, a pH indicating dye. In afurther aspect the reporter can be or include a resazurin dye, atetrazolium dye, coumarin dye, an anthraquinone dye, a cyanine dye, anazo dye, a xanthene dye, an arylmethine dye, a pyrene derivative dye, aruthenium bipyridyl complex dye or derivatives thereof. Certainembodiments utilize a resazurin-based dye. Cell viability dyes, whichare also included in the term reporter used herein, are used as analysisreagents to identify and characterize individual cells or pathogensencapsulated within droplets. Viability dyes have been used since the1950's for cell viability purposes. However, these reagents aretypically employed in samples that are significantly greater than 1microliter in volume and/or are used as an endpoint assay to indicatethe presence of viable cells. Aspects of the invention use a viabilitydye in droplets that are between 1 pL and 100 nL, and more specifically25-500 pL. In the method described here the optical signal generated bythe viability dye is concentrated by the small droplet volume andmeasured and recorded over an incubation time. In droplets containingviable cells, this results in an optical signature that is rapidlygenerated and has information about the characteristics of the cellencapsulated within the droplets. Combined with an environment stressor,such as an antimicrobial or cytotoxic drug, an additional signature canbe generated by monitoring the optical signal of the droplets containinga cell over time. The optical signatures from the cell with and withoutthe environmental stressor can be used to determine the identity and/orcharacteristics of the cell. Furthermore, the differences between theoptical signatures obtained from a species of cells exposed to a drugcompared to the optical signatures for same species of target cells thatare not exposed to the drug can be used to determine the phenotypic drugresistance profile for the target cells obtained from a test sample.Because these signatures are generated from individual cellsencapsulated in droplets, they represent information about theindividual characteristics of each cell as opposed to an averagecharacteristic of a population of cells that is generated from a bulksample containing many cells.

The methods of the invention are compatible with any viability orreporter dye that can be used with live cells (does not require celllysis). In a preferred embodiment the viability dye is a resazurin-baseddye or derivative thereof. When blue, non-fluorescent resazurin isirreversibly reduced to pink and highly fluorescent resorufin itproduces a fluorescent signal and a colorimetric shift (from blue topink). In a preferred embodiment, the fluorescence is used because itoffers better sensitivity over colorimetric signal changes. Thelimited-diffusion confinement within a sub-nanoliter volume of secretedfluorescent molecules quickly concentrates to detectable signal levelsand is then detected by the methods described below. Furthermore,resorufin is reversibly reduced to non-fluorescent hydroresorufin if theredox environment dips below a particular redox threshold, usuallyaround −100 mV. The combination of irreversible reduction from resazurinto resorufin and the reversible reduction of resorufin to hydroresorufinand oxidation of hydroresorufin back to resorufin depending on the redoxpotential of the droplet are what create the unique fluorescencesignature over time in droplets that are small enough volume such thatredox changes occur quickly in the presence of a single cell or cellaggregate. Examples of commercially available resazurin-based dyes are:AlamarBlue™ (various), PrestoBlue™ (Thermo Fisher Scientific),Cell-titer Blue™ (Promega), or Resazurin sodium salt powder. Dyes thatare structurally related to resazurin and can be also be used in themethod are: 10-acetyl-3,7-dihydroxyphenoxazine (also known as AmplexRed™), 7-ethoxyresorufin, and1,3-dichloro-7-hydroxy-9,9-dimethylacridine-2(9H)-one (DDAO dye). Inalternate embodiments dyes that rely on tetrazolium-reduction, such asformazan dyes, can be used as the cell viability indicator. Examplesinclude INT, MTT, XTT, MTS, TTC or tetrazolium chloride, NBT, and theWST series.

Cell (DCC) Aggregates. A preferred application of the invention istowards the diagnosis of microbial infections by identifying themicrobes causing the infection and whether or not they are resistant toantimicrobial drugs. Thus, in this application, the DCCs can besingle-celled microbes. Some bacteria, however, aggregate naturally intoclusters or chains. In these cases, some droplets may comprise anaggregate of cells of the same microbial species (homogenous aggregate)rather than a single microbe. In these cases, the shape of the curve maybe affected by the number of cells in the aggregate. However, the storedsignature waveforms and call logic that are used to classify thecompartmentalized cells can account for such aggregates the same waythey can account for single cells. Furthermore, if the embodimentincludes antimicrobial susceptibility testing the mixture comprising theantimicrobial drug will exhibit the same cell aggregationcharacteristics as the mixture that excludes the antimicrobial drug andthe comparison will still be accurate. Therefore, while the method ofthe invention generally comprises isolation of single-cells in eachdroplet, it necessarily accommodates the case of a single cell speciesin a homogenous aggregate isolated in the droplet rather individualcells. In the case of cancer disease diagnosis, the target DCCstypically do not aggregate if they are circulating tumor cells. If thecancer cells are obtained from tissue, the tissue is typicallydisintegrated into individual cells prior to analysis. Therefore, eachdroplet will contain at most one cell; however, in some instances acancer aggregate may also be analyzed using the described methods.

Signal Detection. Once the droplets have been generated, they must bepresented for analysis by an optical system, sensor, or sensor array. Ina preferred embodiment, the droplets are presented in a 2D array so thatgood thermal control can be maintained, and the droplet signals can bemeasured simultaneously (at a single instance in time) for manydroplets. In the droplets containing target cells, the reporter willproduce a concentrated fluorescent signal that will rise above thebackground droplets that do not contain cells. The concentrated signalof the droplet enables single cell identification in comparable timestandard PCR techniques which are the gold standard for fastidentification. In certain aspects the signal is detected by exciting areduced reporter with a specific wavelength of light and collecting thebandpass-filtered, Stokes-shifted light with a camera. The advantage touse imaging techniques is that they can image a droplet array thatremains stationary and can therefore easily be monitored over time.Cytometry based methods typically employ endpoint detection instead ofreal time detection because of the difficulty in keeping track of themoving droplets over time. Another advantage to imaging the array isthat all the droplets experience the same reaction conditions at thetime of analysis. Therefore, droplet signals can be compared atequivalent time points which is important since signals vary over time.With a cytometry approach, droplets pass by the detector at differenttimes. Therefore, some droplets are incubated longer than others at thetime of analysis. Finally, there may be different target cell species inthe test sample. For each species, there may be an optimal dropletvolume and dye concentration that maximizes signal at a particular timepoint. If an endpoint method is used, droplet volume and reporterconcentrations do not need be controlled to the same degree because timecan compensate for sub optimality and different species can becharacterized universally within a single dye and droplet concentration.

Multiplexing. The methods described herein include the specificidentification of multiple cells from a single test sample. Bycompartmentalizing single cells into their own isolated droplet,competition for resources between cells is eliminated. Therefore,individual cells that would exist collectively as a minority in a bulkpopulation, now have equal access to nutrients when compared to themajority population of cells which results in a higher sensitivity forlow abundance cells in a sample with multiple cells types. Themultiplexing limitations for this invention depend on the ability todifferentiate viability signatures between different cell types. Mostmethods for multiplexing require multiple dyes (fluorophores) which, inturn, require multiple sets of LEDs, excitation, and emission filters.Because the analytical methods use shape information, in some instances,rather than spectral information, the method can be used to multiplexmany targets with a single dye requiring only one LED, emission filter,and excitation filter, thus simplifying the hardware needed to performthe analysis.

E. EXAMPLES

The following examples as well as the figures are included todemonstrate preferred embodiments of the invention. It should beappreciated by those of skill in the art that the techniques disclosedin the examples or figures represent techniques discovered by theinventors to function well in the practice of the invention, and thuscan be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Microchannel fabrication. The droplet array formation experiments wereperformed in cyclic olefin copolymer (COC) microfluidic circuits. Themicrofluidic circuits were fabricated by hot-embossing a COC cover lidfollowed by low temperature bonding with COC film to seal thehot-embossed COC cover lid. The hot embossing master was generated usingphotolithography. The microfluidic circuit geometry is detailed in FIG.1 . The microchannel droplet generator consists of a microchannel thatleads into a well-defined step. The well-defined step then leads into aloosely-defined ramp which in this case is approximated by twoadditional steps.

Reagents and Experimental Protocol. The experiments were performed usinga solution of fluorinated oil with 5% (w/w) PEG-PFPE surfactant as thecontinuous phase. The dispersed phase was a solution of 10% (w/w)resazurin based dye in water. The microfluidic circuit was filled byintroducing 11 μL of the continuous phase to the inlet port. Once thecircuit was filled with the continuous phase the outlet was subsequentlysealed trapping a compressible air bubble between the continuous phaseand the outlet seal. Then 11 μL of the dispersed phase was dispensedinto the inlet port. Care was taken to ensure no air was introducedbetween the dispersed phase and the continuous phase.

The microfluidic circuit was then placed into a sealed system capable ofcontrolling the local pressure while imaging the microfluidic circuit.The system increased the local pressure at a fixed rate to drive thedispersed phase into the microfluidic circuit producing microdroplets ata rate of ˜1 droplet per microchannel droplet generator per second.

An image of the formed droplets was taken (see FIG. 6 ), and thefollowing statistics were computed. Average diameter of droplets=53 μm.Number of droplets=1456. Coefficient of variation on the diameter ofdroplets=2.3%.

1. A microfluidic circuit comprising: (a) a drop forming regioncomprising a channel or nozzle having an inlet for receiving a sample, aconstant cross-sectional area, and an outlet that opens into a stepregion, the channel region or nozzle being rectangular with a height of1 to 20 μm and a width of 2 to 60 μm; (b) the step region having top anda bottom with a height of between 5 to 80 μm, and a substantially flatregion of a length of between 30 to 500 μm extending from the nozzle,the either the top or bottom being substantially continuous with the topor bottom, respectively, of the nozzle and either the bottom or topbeing offset from the bottom or top, respectively, of the nozzle andsubstantially parallel with either the bottom or floor, or top orceiling, respectively, of the nozzle, forming a step region configuredfor finite step emulsification; (c) a ramp region having an angle ofbetween 10 and 80 degrees relative to either the floor or ceiling of thestep region, the ramp region terminates in an imaging region having aheight of 15 to 110 μm; and (d) the imaging region is configured tocollect drops as a two-dimensional array and to provide for imaging andevaluation of the two-dimensional drop array.
 2. The microfluidiccircuit of claim 1, wherein the circuit is fluidly connected to a samplereservoir via a flow path that is configured to provide sample to bereceived by each nozzle inlet.
 3. The microfluidic circuit of claim 2,wherein the circuit is fluidly connected to a waste reservoir configuredto receive sample droplets after analysis.
 4. The microfluidic circuitof claim 1, wherein the ratio of the nozzle height to step region heightis less that 1:3, in particular 1:2.8.
 5. The microfluidic circuit ofclaim 1, wherein the ramp angle is 15 to 45 degrees, particularly 30degrees.
 6. The microfluidic circuit of claim 1, wherein the circuit isthermoplastic polymer.
 7. The microfluidic circuit of claim 1, whereinthe circuit is a cyclic olefin polymer or copolymer, polycarbonate, orpoly (methyl methacrylate) (PMMA).
 8. A microfluidic analysis system,comprising a microfluidic circuit of claim 1 operatively positioned inan image analysis device.
 9. The system of claim 8, wherein the imageanalysis device is configured to detect and analyze fluorescence ofdroplets in the imaging region of the microfluidic circuit.
 10. Thesystem of claim 6, wherein the imaging device comprising a camera and amicrocontroller.
 11. A method of finite step emulsification comprisingflowing a dispersed phase through a nozzle having a constantcross-sectional area into a confined step region containing a continuousphase and forming constrained droplets, providing a ramp region havingan increasing cross-section area through which the constrained dropletsflow forming an unconstrained droplet, where the unconstrained dropletare deposited in an array region and form a two dimensional dropletarray having a low size dispersion.
 12. The method of claim 11, whereinthe dispersed phase is an aqueous sample.
 13. The method of claim 12,wherein the aqueous sample is a biological or environmental sample. 14.The method of claim 11, wherein the continuous is immiscible with thedisperse phase.
 15. The method of claim 14, wherein the continuous phaseis fluorocarbon.